Effects of AT1 receptor antagonism on kainate-induced seizures and concomitant changes in hippocampal extracellular noradrenaline, serotonin, and dopamine levels in Wistar-Kyoto and spontaneously hypertensive rats

YEBEH-04284; No of Pages 6 Epilepsy & Behavior xxx (2015) xxx–xxx

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Effects of AT1 receptor antagonism on kainate-induced seizures and concomitant changes in hippocampal extracellular noradrenaline, serotonin, and dopamine levels in Wistar-Kyoto and spontaneously hypertensive rats Jana Tchekalarova a,⁎, Ellen Loyens b, Ilse Smolders b a b

Institute of Neurobiology, Acad. G. Bonchev Str., Bl. 23, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information, Center for Neurosciences (C4N), Vrije Universiteit Brussel, Brussels, Belgium

a r t i c l e

i n f o

Article history: Received 12 March 2015 Revised 20 March 2015 Accepted 21 March 2015 Available online xxxx Keywords: AT1 receptor antagonist Kainate-induced status epilepticus Monoamines Spontaneously hypertensive rats

a b s t r a c t In the management of epilepsy, AT1 receptor antagonists have been suggested as an additional treatment strategy. A hyperactive brain angiotensin (Ang) II system and upregulated AT1 receptors are implicated in the cerebrovascular alterations in a genetic form of hypertension. Uncontrolled hypertension could also, in turn, be a risk factor for a seizure threshold decrease and development of epileptogenesis. The present study aimed to assess the effects of the selective AT1 receptor antagonist ZD7155 on kainic acid (KA)-induced status epilepticus (SE) development and accompanying changes in the hippocampal extracellular (EC) neurotransmitter levels of noradrenaline (NAD), serotonin (5-HT), and dopamine (DA) in spontaneously hypertensive rats (SHRs) and their parent strain Wistar-Kyoto (WKY) rats, since monoamines are well-known neurotransmitters involved in mechanisms of both epilepsy and hypertension. Status epilepticus was evoked in freely moving rats by a repetitive intraperitoneal (i.p.) administration of KA in subconvulsant doses. In the treatment group, ZD7155 (5 mg/kg i.p.) was coadministered with the first KA injection. Spontaneously hypertensive rats exhibited higher susceptibility to SE than WKY rats, but the AT1 receptor antagonist did not alter the development of SE in SHRs or in WKY rats. In vivo microdialysis demonstrated significant KA-induced increases of the hippocampal NAD and DA levels in SHRs and of NAD, 5-HT, and DA in WKY rats. Although SHRs developed more severe seizures while receiving a lower dose of KA compared to WKY rats, AT1 receptor antagonism completely prevented all KA-induced increases of hippocampal monoamine levels in both rat strains without affecting seizure development per se. These results suggest a lack of direct relationship between KA-induced seizure susceptibility and adaptive changes of hippocampal NAD, 5-HT, and DA levels in the effects of ZD7155 in WKY rats and SHRs. © 2015 Elsevier Inc. All rights reserved.

1. Introduction The predominant role of AT1 receptors in mediating the pathophysiological actions of angiotensin (Ang) II underlies the efficacy and broad use of AT1 receptor antagonists in clinical practice for treatment of hypertension, congestive heart failure, and diabetic nephropathy [1]. Through a blockade of peripheral and central AT1 receptors, the systemic administration of selective AT1 receptor antagonists is able to normalize the increased blood pressure, to alleviate the central and peripheral hormonal and sympathoadrenal responses to stress [2], as well as to exert a neuroprotective effect against ischemia in spontaneously hypertensive rats (SHRs) [3–5]. Besides, accumulating literature data suggest that the AT1 receptor subtype is involved in the modulation of brain excitability, including long-term potentiation and control of seizure ⁎ Corresponding author. Tel.: +359 2979 2172; fax: +359 2 719 109. E-mail address: [email protected] (J. Tchekalarova).

susceptibility [6–10]. Moreover, an upregulation of AT1 receptors in the cortex and the hippocampus of patients diagnosed with temporal lobe epilepsy (TLE) [6] supports the presumption that the effects of Ang II on seizure susceptibility might be mediated by the AT1 receptor subtype. Repetitive seizures induced in an experimental model of TLE caused an upregulation of the angiotensin-converting enzyme and AT1 receptor expression in the hippocampus of Wistar audiogenic rats [8]. Clinically widely used AT1 receptor antagonists have been shown to cross the blood–brain barrier [11,12]. The selective AT1 receptor antagonists have consequently been reported to potentiate the anticonvulsant effect of valproate in mice [7] and to decrease the seizure severity in epileptic rats [8]. Recently, we have found that subchronic systemic infusion of losartan, routinely used as a drug of choice for hypertension, delayed the onset of kainate (KA)-induced status epilepticus (SE) and diminished oxidative stress in SHRs [13]. Chronic hypertension is a strong risk factor for cerebrovascular disease and, thus, predisposing for late-onset seizures and epilepsy in

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Please cite this article as: Tchekalarova J, et al, Effects of AT1 receptor antagonism on kainate-induced seizures and concomitant changes in hippocampal extracellular..., Epilepsy Behav (2015), http://dx.doi.org/10.1016/j.yebeh.2015.03.021

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the elderly [14]. Moreover, the combination of antihypertensive and antiepileptic drugs provides for a relatively safe strategy for treatment of epilepsy associated with hypertension [15]. The SHRs of Okamoto and Aoki [16] have been bred from progenitor Wistar-Kyoto (WKY) rats and used as an experimental research tool for essential hypertension in humans [17]. There is a correlation between deleterious effects of hypertension and abnormalities of the hippocampal structure, neurochemistry, and behavior in SHRs [18–20], suggesting that SHRs may also be explored as a relevant model of comorbidity between hypertension and epilepsy. Only a few studies have been performed to elucidate the role of SHRs in seizure susceptibility [21]. In chronic amygdala and piriform kindling models of epilepsy, SHRs are reported to kindle more rapidly than WKY rats [22]. Spontaneously hypertensive rats exhibited more spontaneous motor seizures than Wistar rats in the KA model of TLE [23]. In addition, whole hippocampal homogenate levels of monoamines and tryptophan were measured but did not differ between epileptic Wistar rats and SHRs [23]. The importance of increased hippocampal monoamine neurotransmission for limiting seizure development has been established earlier [24–26], and therefore, it would be of interest to evaluate possible changes in seizure-evoked hippocampal neurotransmitter dialysate levels in SHRs. In view of the close link between hypertension and epilepsy and the increasing experimental evidence that the broadly used antihypertensive AT1 receptor antagonists might be effective in epilepsy, the major rationale of this study was to further evaluate the effect of the selective AT1 receptor antagonist ZD7155 on the severity of KA-induced SE in a model of essential hypertension and on the extracellular monoamine levels. Compared to the prototypical AT1 antagonist losartan, ZD7155 is a more potent and longer-acting drug [27,28]. In the present in vivo microdialysis study, SE was evoked in freely moving rats by a standard protocol for repetitive intraperitoneal administration of KA in subconvulsant doses. Alterations in hippocampal extracellular noradrenaline (NAD), serotonin (5-HT), and dopamine (DA) efflux were monitored via microdialysis sampling. A close relationship has been reported earlier between the renin–angiotensin system and the central NAD [29,30], 5-HT [31] and DA [31] systems, respectively. Modulation of these neurotransmitter systems could underlie possible effects of AT1 receptor antagonism on KA-induced SE. 2. Materials and methods The procedures used in this study were in agreement with the European Communities Council Directive 2010/63/EU. The experimental procedures were conducted in accordance with national rules on animal experiments and were approved by the Ethics Committee of the Faculty of Medicine and Pharmacy of the Vrije Universiteit Brussel, Belgium. 2.1. Animals Male spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats were purchased from Charles River Laboratories, Brussels, Belgium. The rats were housed in cages having an area of 1650 cm2, and 4 rats were kept in each cage in standardized conditions (12-h/12-h light/dark cycle, lights on at 07:00 h, temperature of 20–23 °C, 50% relative humidity) with free access to food (standard laboratory chow) and water. Rats were habituated in the animal facilities for at least one week. All efforts were made to minimize animal suffering.

diluted in sterile saline (0.9% NaCl) at 2.5 mg/ml. All other chemicals were of analytical reagent grade or better and were supplied by Merck (Darmstadt, Germany). Aqueous solutions were made with purified water (Seralpur pro 90 CN, Belgolabo, Overijse, Belgium) and filtered through a 0.2 μm membrane filter. The perfusion fluid for microdialysis, modified Ringer's solution, contained 147 mM NaCl, 2.3 mM CaCl2, and 4 mM KCl. An antioxidant solution containing 3.3 mM L-cystein, 0.27 Na2EDTA, 12.5 μM ascorbic acid, and 100 mM glacial acetic acid was used to stabilize the collected dialysate to prevent monoamine oxidation. 2.3. Surgery Animals were anesthetized with a mixture of ketamine:diazepam (90:4.5 mg/kg i.p.). Each rat was mounted on a stereotaxic frame for precise intracranial steel guide cannula (CMA Microdialysis, Solna, Sweden) implantation in the left hippocampus as previously described [25]. The coordinates were 4.6 mm lateral and 5.6 mm posterior to the bregma and 4.6 mm ventral starting from the dura [32]. Ketoprofen (4 mg/kg) was administered intraperitoneally (i.p.) for postoperative analgesia. The hippocampal guide cannula obturator was replaced by a microdialysis probe (CMA12; 3-mm membrane length; theoretical cutoff of 20 kDa; CMA Microdialysis, Solna, Sweden). The microdialysis probe was continuously perfused with modified Ringer's solution, and the animal was allowed to recover from surgery overnight before the experimental procedure. 2.4. Experimental design The microdialysis study design is described in Fig. 1. Animals were randomly divided into four main groups: WKY rats treated with KA and saline (WKY/KA), WKY rats treated with KA and ZD7155 (WKY/KA + ZD7155), SHRs treated with KA and saline (SHRs/KA), and SHRs treated with KA and ZD7155 (SHRs/KA + ZD7155). The microdialysis probe was continuously perfused with modified Ringer's solution at a flow rate of 2 μl/min. Individual sampling times were set at 20 min. Each experiment started with the collection of six samples in which the perfused fluid was composed of modified Ringer's solution (2 μl/min) followed by six samples composed of modified Ringer's solution and obtained after two i.p. injections of saline (1 ml/kg) with an interval of 1 h (Fig. 1). This protocol for the collection of samples before the start of KA treatment was conducted to adapt animals to the successive i.p. injections. Consecutively, different protocols were applied in which a single i.p. injection of either saline or ZD7155 (5 mg/kg) was concomitantly applied with the first injection of KA (5 mg/kg i.p.). It has been shown that the oral administration of 3 mg/kg ZD7155 to Sprague–Dawley rats results in a rapidly lowered blood pressure for up to 48 h (IC50 value = 3.8 nM in guinea pig adrenal gland membranes) [28]. The treatment with KA continued until sustained convulsive seizures of classes 3–5 (i.e., N9 motor seizures per hour) were observed in rats. The number of motor seizures was registered and used as criteria for an additional KA injection. Every hour, the severity of seizures and well-being of the rat were assessed to determine whether the next dose of KA should be (a) full (5 mg/kg), (b) half (2.5 mg/kg), or (c) omitted. Seizure severity was evaluated by a modified Racine's scale [33] described earlier [23]. The whole period of observation after the first KA injection was 140 min. Seizure severity was determined by the summation of the highest scores obtained during each microdialysis collection period, resulting in a total seizure severity score (TSSS) for each animal. 2.5. Microdialysate analysis

2.2. Drugs and reagents The neurotoxin kainic acid (KA) was purchased from Ascent Scientific (UK). The selective AT1 receptor antagonist ZD7155 was supplied by Tocris Cookson Ltd. (Bristol, UK). Kainic acid and ZD7155 were

For monoamine analysis, an isocratic microbore liquid chromatography (LC) assay (C8, 5 μm; 100 × 1 mm; Unijet, Bioanalytical Systems, West Lafayette, IN, USA) coupled to amperometric detection (Decade, Antec, Leiden, the Netherlands) was used as previously described [34].

Please cite this article as: Tchekalarova J, et al, Effects of AT1 receptor antagonism on kainate-induced seizures and concomitant changes in hippocampal extracellular..., Epilepsy Behav (2015), http://dx.doi.org/10.1016/j.yebeh.2015.03.021

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2/ calculation of TSSS within this 140 min time period 3/ calculation of the dialysate content of NAD, 5-HT, and DA within this 140 min time period for each experimental rat group receiving KA with or without ZD7155

2/ verifying possible interference of repeated i.p. saline (sham) injections (during collection 7 and collection 10)

Fig. 1. Schematic illustration of the experimental protocol.

3. Results 3.1. Effect of ZD7155 on kainate-induced status epilepticus in WKY rats and SHRs Both normotensive WKY rats and SHRs demonstrated typical behavioral changes starting with wet-dog shakes, excessive grooming, and increased exploratory behavior 20–30 min after the first injection of 5 mg/kg KA. During the second hour of the observation when an additional dose of 5 mg/kg KA was injected, these changes evolved into recurrent bouts of staring and mouth and facial movements. Within 30 min, these excitatory features progressively developed into intermittent limbic motor seizures characterized by facial muscle clonus, forelimb clonus, and rearing in SHRs. In most of the WKY rats, limbic motor seizures appeared during the second half of the 2nd hour. During the next 60 to 90 min, seizures gradually increased to sustained SE with recurrent seizures (stage 3/4/5) in all rats. Spontaneously hypertensive rats were characterized by a higher susceptibility to KA than WKY rats, with a lower total dose of KA required to induce SE and higher mean TSSS [Genotype effect: F1,32 = 16.241, p b 0.001; Holm–Sidak post hoc test: SHRs/KA vs. WKY/KA, p b 0.05] (Figs. 2A, B). The selective AT1 receptor antagonist ZD7155 failed to protect WKY rats and SHRs against the development of KA-induced SE. The dose of KA required to induce SE and the mean TSSS did not differ between saline-treated and ZD7155-treated rat groups (p N 0.05) (Figs. 2A, B). 3.2. Effects of ZD7155 on kainate-induced changes in hippocampal monoamine levels 3.2.1. NAD No significant differences in the EC NAD baseline concentrations between WKY rats and SHRs were detected (Figs. 3A, B). There was

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The TSSSs and the extracellular (EC) concentrations of NAD, 5-HT, and DA (calculated in nM) in baseline conditions and after the pharmacological manipulation are represented as means ± SEM. The experimental data were evaluated by two-way ANOVA (factors Genotype × Treatment (KA or ZD7155)) followed by the post hoc Holm–Sidak test. For all statistical analysis, α was set at 0.05.

also no effect of repetitive i.p. saline injections on the EC NAD levels during the baseline collections (data not shown). In the rats treated with KA and a saline sham injection, two-way ANOVA showed a main

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Fig. 2. (A) Dose of KA required to induce status epilepticus (SE) in normotensive WistarKyoto (WKY) rats and spontaneously hypertensive rats (SHRs). +p b 0.05 vs. respective WKY groups. (B) Clinical seizure severity represented by KA-induced total seizure severity score (TSSS) in WKY rats and SHRs treated with saline (WKY/KA and SHRs/KA) and the selective AT1 receptor antagonist ZD7155 (5 mg/kg i.p.) (WKY/KA + ZD and SHRs/KA + ZD). + p b 0.05 vs. respective WKY groups.

Please cite this article as: Tchekalarova J, et al, Effects of AT1 receptor antagonism on kainate-induced seizures and concomitant changes in hippocampal extracellular..., Epilepsy Behav (2015), http://dx.doi.org/10.1016/j.yebeh.2015.03.021

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effect of the Treatment factor (KA) on the EC NAD concentration [F1,32 = 11.363, p b 0.002], but neither the overall Genotype nor the Genotype × Treatment interaction showed significant differences. The KA injection elicited a statistically significant increase of the mean NAD dialysate concentration during the 140 min sampling period both in WKY rats (p = 0.018) and in SHRs (p = 0.006), respectively (Fig. 3A). In the rats receiving both KA and ZD7155, two-way ANOVA did not reveal any effects of Treatment or Genotype on the EC NAD concentrations (Fig. 3B). Pretreatment with ZD7155 prevented the KA-induced increase in the EC NAD concentration in WKY rats (Fig. 3B). 3.2.2. 5-HT Baseline hippocampal 5-HT levels were not significantly different between WKY rats and SHRs (Figs. 4A, B). There was no effect of repetitive i.p. saline injections on the EC 5-HT levels during the baseline collections (data not shown). In rat groups treated with KA and a saline sham injection, two-way ANOVA revealed a main effect of Treatment [F1,25 = 5.777, p b 0.025] and Genotype [F1,25 = 6.209, p b 0.021] on the EC 5-HT concentration without the Genotype × Treatment interaction. In KA-treated WKY rats, the mean 5-HT dialysate concentration was significantly elevated (*p = 0.03) (Fig. 4A). Furthermore, a significant difference between WKY rats and SHRs was detected after KA treatment (p = 0.002). Kainic acid did not produce significant changes in the hippocampal EC 5-HT levels in SHRs (p N 0.05). In the rat groups receiving both KA and ZD7155, there were no significant effects on hippocampal 5-HT levels (Fig. 4B). The AT1 receptor antagonist attenuated the KA-induced increase in the EC 5-HT levels in WKY rats.

SHR n=7 n=7

Fig. 4. Effect of KA (A) and ZD7155 (B) on hippocampal EC concentrations of serotonin 5-HT. Other details as in Fig. 3. *p b 0.05 vs. basal neurotransmitter levels; op b 0.05 vs. WKY rats treated with KA.

on the EC DA levels during the baseline collections (data not shown). In rat groups receiving KA and a saline injection, a main effect of Treatment [F1,29 = 11.148, p b 0.003] without the overall Genotype and the Genotype × Treatment interaction was found. The mean total hippocampal EC DA concentration was markedly elevated both in KA-treated WKY rats (p b 0.045) and in SHRs (p b 0.015), respectively (Fig. 5A). The injection of ZD7155 prevented the KA-induced hippocampal efflux of DA in WKY rats and SHRs since no significant effects were found in both rat strains treated with KA and ZD7155 injections (Fig. 5B).

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Fig. 3. Effect of KA (A) and ZD7155 (B) on hippocampal EC concentrations of noradrenaline (NAD). The mean neurotransmitter levels during baseline collections (0–120 min) and saline-treated dialysate collections (140–240 min) (basal) were compared with the mean neurotransmitter levels during the seven collection periods after the start of KA treatment (collection periods of 260–380 min) (KA) (A) and the mean neurotransmitter levels during the seven collection periods after ZD7155 injection and the start of KA treatment (collection periods of 260–380 min) (KA + ZD7155) (B). *p b 0.05 vs. basal neurotransmitter levels.

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3.2.3. DA No significant differences in the baseline hippocampal DA dialysate concentrations were detected between WKY rats and SHRs (Figs. 5A, B). There was no effect of repetitive i.p. saline injections

WKY n=7 n=7

SHR n=7 n=7

Fig. 5. Effect of KA (A) and ZD7155 (B) on hippocampal EC concentrations of dopamine. Other details as in Fig. 3.

Please cite this article as: Tchekalarova J, et al, Effects of AT1 receptor antagonism on kainate-induced seizures and concomitant changes in hippocampal extracellular..., Epilepsy Behav (2015), http://dx.doi.org/10.1016/j.yebeh.2015.03.021

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4. Discussion The present study demonstrated for the first time, to our knowledge differences between SHRs and their parent strain normotensive WKY rats in seizure susceptibility in the KA-induced SE model and in the KAevoked seizure-associated changes of monoamine efflux in the hippocampus. In WKY rats and in SHRs, AT1 receptor antagonism with ZD7155 abolished all seizure-associated increases in hippocampal monoamine levels but did not alter KA-induced seizure severity per se. The SHRs exhibited higher seizure susceptibility than WKY rats as illustrated by a lower dose of KA needed to elicit SE and a higher TSSS. Although no significant differences in the average dose of KA required to induce SE in Wistar rats and SHRs were found earlier, SHRs have already been characterized by a higher seizure frequency than Wistar rats during the chronic epileptic phase [23]. Spontaneously hypertensive rats were also more sensitive to tonic extensor seizures [21] and to electroshock-induced convulsions [35] compared to Wistar rats as well as to amygdala or cortical kindling stimulation compared to WKY rats [22]. Our previous experimental findings revealed that SHRs might be used as a model of attention-deficit hyperactivity disorder (ADHD) and epilepsy (reviewed in [36]), confirming clinical data for a bidirectional link between epilepsy and ADHD (reviewed in [37]). In addition to hyperactivity, attention deficit, and impulsiveness, SHRs demonstrated biochemical changes in 5-HT [38] and DA neurotransmission in the frontal cortex compared to their controls [39]. To the best of our knowledge, our present study is the first to demonstrate that systemically injected KA increases all hippocampal monoamine dialysate levels in WKY rats and NAD and DA levels in SHRs, although increases in hippocampal EC GABA and glutamate levels following KA have been monitored on several occasions in other rat strains and structures [40–44]. The WKY rats were more resistant than SHRs to the development of KA-induced SE but, unlike SHRs, showed a significant increase in the hippocampal 5-HT levels after KA injection. Previous studies considering the effect of seizures on hippocampal EC 5-HT have demonstrated conflicting results: some authors reported an increase in the hippocampal 5-HT overflow [45,46] while others found no seizure-associated changes [25,47], supporting that 5-HT alterations in the hippocampus can be strain- and model-dependent. Our present study shows that KA-induced SE can provoke an increase in hippocampal 5-HT in WKY rats, therefore supporting the established anticonvulsant role of increased hippocampal 5-HTergic neurotransmission [24,26,48] in WKY rats. The hippocampal NAD overflow and DA overflow were increased both in SHRs and in WKY rats during KA-induced SE, confirming previous observations with other seizure models and strains [25,45,49–51]. These findings suggest a common mechanism accompanying seizure activity which triggers NAD and DA release in the hippocampus rather than specific pharmacological effects of different chemoconvulsants in different rat strains. Extensive evidence supports the regulatory role of NADergic neurotransmission in the control of seizure susceptibility, including seizure propagation in the limbic structures [52,53]. Genetically epilepsy-prone rats (GEPR), in which the NADergic system has been disturbed, are more sensitive to experimentally evoked seizures, and restoration of NADergic activity has been shown to attenuate the enhanced seizure susceptibility [54,55]. Moreover, activation of the locus coeruleus NADergic system [56] and hippocampal NAD reuptake inhibition [57] is able to alleviate seizure activity induced by chemoconvulsants. Despite the observed seizure-evoked increase in the DA levels measured by microdialysis, we have recently reported a decrease in the total DA hippocampal homogenate content in both SHRs and Wistar rats during the chronic epileptic state in a model of epilepsy and depression comorbidity [23]. Thereby, while the compromised DA neurotransmission might reflect specific behavioral responses linked to a depressivelike state and enhanced spontaneous seizure activity during the chronic

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phase of epilepsy, the increased EC DA concentration during SE per se may represent an adaptive mechanism limiting further the development of seizure activity. A final question raised in our work was whether the blockade of AT1 receptors was able to protect SHRs, which are characterized by an activated renin–angiotensin system, against KA-induced SE. Although the dose of 5 mg/kg ZD7155 was shown to be effective in lowering the arterial blood pressure in hypertensive rats [28], it was ineffective in attenuating the KA-induced seizure severity. Recently, chronically infused losartan did not prevent the development or severity of KA-induced SE but increased the latency for the onset of status epilepticus [13]. Furthermore, losartan attenuated the KA-induced increase in lipid peroxidation both in SHRs and in Wistar rats and produced higher expression of heat shock protein 72 in the hippocampus [13]. Chronic high-dose treatment with losartan (50 mg/kg for 21 days) has also been reported to prevent limbic seizures in a model of TLE [8]. Therefore, long-term treatment rather than a single injection could be more effective in the prevention of the seizure-related consequences of SE. Numerous in vitro and in vivo results provide evidence that both peripheral and central AT1 receptors are involved in NAD and DA neuromodulation. Thus, the blockage of the presynaptic [3H]-NAD release by chronic treatment with losartan in isolated atria of SHRs was suggested to participate in the antihypertensive action of the AT1 receptor antagonists [58]. Furthermore, the Ang II-stimulated striatal DA release was found not to be significantly different between SHRs and WKY rats [59], and in normotensive rats, the Ang II-induced increase in striatal DA release was completely antagonized by losartan [60]. In SHRs, the selective AT1 receptor antagonist CV 11974 decreased arterial pressure, an effect accompanied by a significant increase in the release of GABA in the rostral ventrolateral medulla [61]. There are no literature data of the effect of AT1 receptor antagonism on monoamine release in the hippocampus. Our results, therefore, demonstrate for the first time that acute injection of 5 mg/kg ZD7155 completely abolished the KA-evoked increases in elevated hippocampal NAD, 5-HT, and DA concentrations in WKY rats and 5-HT concentrations in SHRs. In summary, the present study demonstrated differences between SHRs and their parent strain normotensive WKY rats in seizure susceptibility in the KA-induced SE model and in the KA-evoked seizureassociated changes of monoamine efflux in the hippocampus. In both rat strains, AT1 receptor antagonism with ZD7155 abolished all KAassociated increases in hippocampal monoamine levels without altering KA-induced seizure development, suggesting a lack of a link between KA-induced seizure susceptibility and adaptive changes of hippocampal NAD, 5-HT, and DA in the effects of ZD7155 in WKY rats and SHRs.

Acknowledgments This study was supported by a Joint Research Project between the Fund for Scientific Research (FWO)—Flanders (Belgium) and the Bulgarian Academy of Sciences (2010–2012) (grant no. VS.039.10N). Disclosure/conflicts of interest None of the authors has any conflict of interest concerning this manuscript.

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Please cite this article as: Tchekalarova J, et al, Effects of AT1 receptor antagonism on kainate-induced seizures and concomitant changes in hippocampal extracellular..., Epilepsy Behav (2015), http://dx.doi.org/10.1016/j.yebeh.2015.03.021